An unusual Cretaceous beetle with affinity to Anamorphidae (Coleoptera: Coccinelloidea)

Coccinelloid beetles have a sparse fossil record in the Mesozoic. Here, we describe and illustrate an unusual coccinelloid beetle, Yassibum yoshitomii gen. et sp. nov., from mid-Cretaceous Kachin amber. Yassibum stands out within the Coccinelloidea due to its notched profemora and the presence of antennal grooves on the elytral epipleura. Based on our phylogenetic analyses, we suggest that Yassibum is most likely related to the family Anamorphidae. The alternative placements are critically evaluated based on our comparison of the morphology.


Materials
The Kachin (Burmese) amber specimen studied herein (Figs.1-5) originated from amber mines near Noije Bum (26 20′N, 96 36′E), Hukawng Valley, Kachin State, northern Myanmar, and is deposited in the Nanjing Institute of Geology and Palaeontology (NIGP), Chinese Academy of Sciences, Nanjing, China.The amber piece was trimmed with a small table saw, ground with emery papers of different grit sizes, and finally polished with polishing powder.Jewellery-grade Kachin amber specimens are commonly carried and sold legally in Ruili, Dehong Prefecture on the border between China and Myanmar.The specimen in this study was purchased in 2016 (prior to 2017), and was therefore not involved in the armed conflict and ethnic strife in Myanmar.

Fossil imaging
Photographs under incident light were taken with a Zeiss Discovery V20 stereo microscope.Widefield fluorescence images were captured with a Zeiss Axio Imager.Z2 light microscope combined with a fluorescence imaging system.Confocal images were obtained with a Zeiss LSM710 confocal laser scanning microscope, using the 561 nm (DPSS 561-10) laser excitation line (Fu et al., 2021).Images under incident light and widefield fluorescence were stacked in Helicon Focus 7.0.2 and Zerene Stacker 1.04.Confocal images were stacked with Helicon Focus 7.0.2 and Adobe Photoshop CC.Microtomographic data were obtained with a Zeiss Xradia 520 Versa 3D X-ray microscope at the micro-CT laboratory of NIGP and analyzed in VGStudio MAX 3.0.Scanning parameters were as follows: isotropic voxel size, 1.6339 mm; power, 3 W; acceleration voltage, 40 kV; exposure time, 4 s; projections, 3001.Images were further processed in Adobe Photoshop CC to adjust brightness and contrast.

Phylogenetic analyses
To evaluate the systematic placement of the new fossil, we conducted constrained morphology-based phylogenetic analyses under parsimony (e.g., Li et al., 2023aLi et al., , 2023b)).During the constrained analyses, the relationships among extant taxa were fixed as the backbone tree according to previous molecular studies, and only the fossil was allowed to move freely.The use of molecular-based constraints would allow a more realistic estimation of the states at ancestral nodes, and therefore contribute to a more authentic placement of the fossil (Fikáček et al., 2020).2021).The position of Loeblioryloninae (Loebliorylon Ślipi nski) has not been studied with molecular data and is largely unclear; therefore it was not included in the backbone tree of our analysis.
The analyses were performed under implied weights, using R 4.1.0(R Core Team, 2021) and the R package TreeSearch 1.3.1 (Smith, 2023).The concavity constant was set to 12, following the suggestion by Goloboff, Torres & Arias (2018) and Smith (2019).In order to inspect both the optimal and suboptimal placements of the fossil, the parsimony scores of the trees with alternative placements of the fossil were mapped to the corresponding branches of the backbone tree (Li et al., 2024a(Li et al., , 2024b)).The results were visualized with the R package ggtree 6.5.2 (Yu et al., 2017;Yu, 2020) and graphically edited with Adobe Illustrator CC 2017.

Nomenclature
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone.This published work and the
Etymology.The species is named after the coleopterist Dr. Hiroyuki Yoshitomi.
Diagnosis.As for the genus.
Pronotum transverse, laterally slightly explanate; sides gradually converging anteriorly in anterior half; lateral pronotal carinae complete, weakly denticulate; posterior margin broadly and weakly produced backwards medially; anterior angles not produced; disc with narrow raised margins, with a pair of curved narrow basolateral sulci.Prosternal process short, reaching only middle of procoxae.Procoxae longer than wide, strongly projecting.
Scutellar shield small, transverse, anteriorly abruptly elevated.Elytra completely covering abdomen, widest near anterior 1/4, gradually narrowing posteriorly, laterally widely explanate; sides rounded; disc irregularly punctate, with long parasutural stria extending from elytral apex to near scutellum; epipleura wide at base, incomplete towards apices, each basally with well-developed posterolaterally extending antennal groove and anteriorly directed projection near base of antennal groove.Mesoventrite longitudinally carinate at middle, posteriorly bifid, without visible pits or pores.Mesocoxal cavities separated by almost diameter of cavity, laterally broadly closed by meeting of meso-and metaventrites.Metaventrite large, with raised margin anteriorly, with distinct depression at each anterolateral corner (likely for accommodating mesotrochanter), without visible pits or pores near anterior margin.Metanepisterna concealed.

DISCUSSION
Yassibum gen.nov.can be doubtlessly assigned to Coccinelloidea based on its oval and convex body, reduced tarsomeres (4-4-4), and widely separated metacoxae with truncate intercoxal process (Robertson et al., 2015).The strongly reduced hindwing venation is also accordant with the coccinelloid placement (Lawrence et al., 2022).As the character combination of Yassibum does not seem to perfectly fit with any of the existing coccinelloid families, we conducted constrained parsimony analyses under implied weights to test its familial assignment.In the analysis based on the matrix developed by Lawrence et al. (2011), Yassibum is best placed in Anamorphidae, whereas Alexiidae and Endomychidae appear to be the suboptimal alternative placements.If not considering the possibilities of being inserted to the interfamilial branches, the analysis based on the matrix by Robertson (2010) produced overall similar results, with Anamorphidae, Latridiidae, Alexiidae and Endomychidae being the most parsimonious placements for Yassibum.
The concealed metanepisternum is an unusual character for Yassibum, which is known in Discolomatidae and some Latridiidae (Lawrence et al., 2011).In the analysis based on the matrix by Robertson (2010), the topology also receives a low parsimony score when Yassibum is inserted to the ancestral branch of Latridiidae.However, Yassibum is unlikely to belong to either Discolomatidae or Latridiidae.Discolomatidae differ from Yassibum in the antennae with one-segmented club, presence of gland openings along lateral pronotal and elytral margins, externally small and rounded metacoxae, and abdomen with five ventrites (John, 1959;Cline & Ślipi nski, 2010;Cline & Shockley, 2012;Yoshitomi & Pham, 2021).Latridiidae differs from Yassibum in the elongate body shape, 3-3-3 tarsi and often regularly punctuate elytra (Hartley & McHugh, 2010;Lord et al., 2010;Chan & Lee, 2016).
The dorsally notched profemur is notable for Yassibum.While in many members of Anamorphidae or Endomychidae the fore, mid or hind legs are notched or toothed, these modifications occur exclusively on the ventral face of the trochanter, femur or tibia (e.g., Tomaszewska, 2000aTomaszewska, , 2000b)).The procoxae are prominent (associated with the vestigial procoxal process) in some Endomychidae (e.g., Archipines Strohecker or Hylaperdina Tomaszewska; Arriaga-Varela & Tomaszewska, 2016: fig.10).However, they are not so strongly projecting like in Yassibum.The position and possible use of the modification on the elytral epipleuron is another peculiar feature for Yassibum.At the base of elytral epipleuron, there is a posterolaterally extending groove, and near the base of this groove there is an anteriorly directed projection.Based on the position and shape of this groove, we suppose that it is likely to be used to accommodate the antenna, rather than the profemur.Antennal grooves are commonly found on the head or prothorax of beetles, however, as far as we know, this is the first time for the antennal groove to be discovered on the elytral epipleuron in the whole Coleoptera.A somewhat similar groove on elytral epipleuron is known in the extinct archostematan genus Notocupes Ponomarenko, but in that case it is used for accommodating the mid leg (Li et al., 2023c(Li et al., , 2023d)).
Yassibum clearly differs from the other anamorphid beetles known from the mid-Cretaceous, Palaeosymbius spp.(Arriaga-Varela et al., 2024), by the emarginate labrum, profemora with an incision in the exterior margin, mesoventrite without visible pits or pores and with posterior margin of intercoxal process bifid, elytral epipleura excavated, possibly for the reception of the antenna, and metaventrite with anterior margin without pores or pits.likely affinity with extant genera Symbiotes and Asymbius, the possible position of Yassibum within the anamorphid lineages is yet to be fully understood.Although the overall shape of the pronotum could suggest a resemblance to genera like Bystus and Bystodes, the peculiar characters of the genus described here point out the possibility of a lineage within Anamorphidae that left no descendants in present time.

Figure 1 Figure 2
Figure 1 General habitus of Yassibum yoshitomii gen.et sp.nov., holotype, NIGP200730, under incident light.(A) Dorsal view.(B) Ventral view.Scale bars: 500 mm.Full-size  DOI: 10.7717/peerj.18008/fig-1 We applied two matrices to evaluate the phylogenetic position of the new fossil, one based on Lawrence et al. (2011) (Data S1), and the other based on Robertson (2010) (Data S2).The matrix by Lawrence et al. (2011) had broad sampling among the whole Coleoptera, but in the present analysis we only selected the coccinelloid genera and a few cucujiform outgroups.The matrix by Robertson (2010) was focused on Coccinelloidea with higher density of taxon sampling, but had fewer morphological characters.For the new fossil, we successfully coded 134 characters with non-inapplicable states in the matrix based on Lawrence et al. (2011), and 44 characters in the matrix based on Robertson (2010).
For the construction of backbone trees, the interfamilial relationships were mainly based on McKenna et al. (2019) and Cai et al. (2022), the positions of Akalyptoischiidae, Mycetaeidae and Eupsilobiidae were based on Arriaga-Varela et al. (2023b), and the intrafamilial relationships not covered by the above studies were mainly based on Robertson et al. (2015) and Che et al. (

Figure 7
Figure 7 Constrained parsimony analysis under implied weights, based on the matrix by Robertson (2010), showing alternative placements of Yassibum gen.nov.The score above each branch represents the parsimony score of the topology in which Yassibum is inserted to that branch.The colors are an intuitive display of the parsimony scores.Full-size  DOI: 10.7717/peerj.18008/fig-7 Etymology.The generic name is an anagram of Asymbius Gorham, a genus in Anamorphidae.The name is neuter in gender.Diagnosis.Body oval.Antennae 10-segmented, with 3-segmented club.Labrum with apex deeply emarginate.Pronotal disc with narrow and deep basolateral sulci.Procoxae distinctly projecting.Elytral epipleura wide at base, basally with antennal groove extending posterolaterally.Mesocoxal cavities broadly closed laterally.Mesoventrite and metaventrite without pits near their anterior margins.Metanepisterna concealed.Profemora with prominent incision on outer side.Tarsi simple, 4-4-4.
While the characters of Palaeosymbius Arriaga-Varela et al. show a veryFigure 6 Constrained parsimony analysis under implied weights, based on the matrix by Lawrence et al. (2011), showing alternative placements of Yassibum gen.nov.The score above each branch represents the parsimony score of the topology in which Yassibum is inserted to that branch.The colors are an intuitive display of the parsimony scores.